The objective of this application is to uncover roles for RNA structure in regulating biological activity of the RNA-activated protein kinase, PKR. Viral and cellular RNAs fold into diverse secondary and tertiary structures and interact with proteins to alter the innate immune response. A key player in innate immunity is the interferon-induced RNA-activated protein kinase, PKR. The major activator of PKR in vivo has been proposed to be long dsRNA (>33 bp), which can bridge two PKR monomers and increase their effective concentration. Interaction with dsRNA also relieves an inhibitory interaction in the kinase. Subsequently, PKR carries out trans-autophosphorylation that activates it to phosphorylate eIF21, which inhibits the initiation of translation. Despite the obvious ability of dsRNA to activate PKR in vitro, it remains unclear whether such RNAs are major activators of PKR in vivo. The central hypothesis of this proposal is that regulation of PKR in vivo is mediated by novel RNA motifs with unconventional structures. In particular, evidence is presented that ssRNA activates PKR in a 5'-triphosphate-dependent fashion, which is blocked by cellular 5'-end signatures of 7mG and monophosphate. Because a 5'-triphosphate occurs on many pathogenic RNAs, this suggests a novel pathogen-associated molecular pattern (PAMP) that is recognized by PKR. Moreover, additional evidence suggests that self RNA is also distinguished by internal nucleoside modifications, which are shown to abrogate PKR activation. The central aims of the proposal are as follows: 1.) Determine the mechanism by which ssRNA activates PKR in a 5'-triphosphate-specific fashion, often with the assistance of short stem-loops. Develop a molecular model for interaction of the 5'-triphosphate and short stem-loop with PKR, as well as a mechanistic framework for activation. 2.) Establish roles for posttranscriptional RNA modifications and non-Watson-Crick motifs in modulating PKR activation. Identify patterns of modifications and non-Watson-Crick motifs that allow cellular RNAs to evade PKR activation. 3.) Determine roles for short viral RNA secondary structures and globular tertiary motifs in modulating PKR activation. Test if certain viral secondary structure RNAs dimerize to form PKR-activating motifs, while globular RNA tertiary structures fold to mask PKR-activating RNA secondary structures. 4.) Identify viral and endogenous RNAs that regulate PKR in vivo. Use cross-linking and immunoprecipitation (CLIP) technologies with (and without) vesicular stomatitis virus transfections and infections. These Specific Aims will be accomplished by a variety of biochemical and molecular biology techniques including RNA and protein mutagenesis, in vitro and in vivo PKR and eIF21 activation assays, kinetics and thermodynamic measurements, and CLIP experiments. Binding assays will be conducted by fluorescence polarization and ITC;protein dimerization will be monitored by pull-down assays, crosslinking, and analytical ultracentrifugation;RNA-protein interactions will be assayed by chemical crosslinking and mutagenesis;and RNA tertiary structure will be assessed by native gel electrophoresis and structure mapping. PUBLIC HEALTH RELEVANCE: Innate immunity offers a host early protection from foreign organisms and viruses, and the protein PKR is an important part of this response in humans. This proposal aims to understand how novel molecular patterns in pathogenic RNA are recognized by PKR as different from self RNA, which cause the innate immune response to be initiated. Viral RNAs with potentially pathogenic patterns from human immunodeficiency virus (HIV) and hepatitis delta virus (HDV) will be studied.